Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology · 2007
[Construction and screening of the specific Fab phage antibody library against Raji cell strain].
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Xie Xiao-fang, Shen Yong-mei, Yang Xiao-chun, Dong Ning-zheng, Bai Xia, Shi Yi-zhen
Аннотация
AIM: To construct and screen the specific Fab phage antibody library against human Raji cell strain in B-lymphoma.
METHODS: BALB/c mice were immunized with Raji cells, and the antibody light chain kappa genes and heavy chain genes Fd from the spleen cells were amplified by RT-PCR. After restrictive digestion with Sac I/Xba I and Xho I/Spe I, the light chain kappa genes and heavy chain genes Fd were inserted into the phagemid vector pComb3H-SS successively and then electroporated into E.coli XL1-Blue. The specific Fab phage antibody library against Raji cell strain in human B-lymphoma was constructed by infection of helper phage VCSM13. The specific antibodies against Raji cells were obtained after selected with Raji cells. The binding activity with antigens was identified by ELISA and the positive clones were sequenced.
RESULTS: The Fab phage antibody library with 2.18 x 10(7) volume was constructed and eight positive clones which specifically recognized Raji cell strain were isolated. Sequence analysis of the two positive clones showed that the variable heavy domains (VH) and variable light domains (VL) were highly homologous with the registered murine Ig heavy chain V region sequences and kappa light chain sequences, respectively.
CONCLUSION: Fab phage antibody library was successfully constructed and specific antibodies against membrane antigens in Raji cells were obtained, which will provide an experimental foundation for the further investigation of B-lymphoma immunotherapy.
METHODS: BALB/c mice were immunized with Raji cells, and the antibody light chain kappa genes and heavy chain genes Fd from the spleen cells were amplified by RT-PCR. After restrictive digestion with Sac I/Xba I and Xho I/Spe I, the light chain kappa genes and heavy chain genes Fd were inserted into the phagemid vector pComb3H-SS successively and then electroporated into E.coli XL1-Blue. The specific Fab phage antibody library against Raji cell strain in human B-lymphoma was constructed by infection of helper phage VCSM13. The specific antibodies against Raji cells were obtained after selected with Raji cells. The binding activity with antigens was identified by ELISA and the positive clones were sequenced.
RESULTS: The Fab phage antibody library with 2.18 x 10(7) volume was constructed and eight positive clones which specifically recognized Raji cell strain were isolated. Sequence analysis of the two positive clones showed that the variable heavy domains (VH) and variable light domains (VL) were highly homologous with the registered murine Ig heavy chain V region sequences and kappa light chain sequences, respectively.
CONCLUSION: Fab phage antibody library was successfully constructed and specific antibodies against membrane antigens in Raji cells were obtained, which will provide an experimental foundation for the further investigation of B-lymphoma immunotherapy.